The ability of microbial pathogens to overcome the normally highly polarized host mucosal epithelial barrier is an early and critical step in pathogenesis. This is particularly critical for opportunistic pathogens such as Pseudomonas aeruginosa (PA), one of the most virulent opportunistic pathogens of man. In the setting of epithelial injury and loss of cell polarity, however, PA can effectively colonize the mucosal surfaces and cause further damage, prevent repair of the wounded epithelium, and disseminate. Our long term goals are to understand how pathogens in general, and PA in particular, overcome the host epithelial barrier to cause human disease. Our short term goals are to understand how PA hijacks host signaling pathways to enter into cells and how disruption of cell polarity predisposes to PA invasion. We have used a novel forward genetic approach, genome-wide RNAi-mediated gene inactivation, to carry out a targeted genetic screen to identify host proteins important for bacterial binding and internalization. From this screen, we have identified many new host genes required for entry of strain PAK, including a putative receptor (E-cadherin), adaptor proteins (Abl/arg kinase and Crk), regulators of the cytoskeleton (Cdc42, Rac, and p21-activated protein kinase (Pak)), and downstream effectors (phosphoinositol-3-phospho kinase (PI3K) and Akt). We are uniquely poised now to determine how these host molecules are subverted by PA when it enters mammalian cells and whether it is relevant to human disease. These studies, which employ novel genetic approaches and state-of-the-art cell biology, will comprehensively dissect the interactions between PA and host cell epithelium. They will identify host factors that the bacteria exploit to cause disease. These host cell factors may serve as novel targets for the development of anti-bacterial therapeutics; because the drug targets host but not bacterial molecules, they are much less likely to engender resistance compared to conventional anti-microbial therapies.